Reproductive toxicity assessment of cellulose nanofibers, citric acid, and branched polyethylenimine in sea urchins: Eco-design of nanostructured cellulose sponge framework (Part B).

In the framework of a safe-by-design approach, we previously assessed the eco-safety of nanostructured cellulose sponge (CNS) leachate on sea urchin reproduction. It impaired gamete quality, gamete fertilization competence, and embryo development possibly due to the leaching of chemical additives used during the CNS synthesis process. To extend this observation and identify the component(s) that contribute to CNS ecotoxicity, in the present study, we individually screened the cytotoxic effects on sea urchin Arbacia lixula and Paracentrotus lividus gametes and embryos of the three main constituents of CNS, namely cellulose nanofibers, citric acid, and branched polyethylenimine. The study aimed to minimize any potential safety risk of these components and to obtain an eco-safe CNS. Among the three CNS constituents, branched polyethylenimine resulted in the most toxic agent. Indeed, it affected the physiology and fertilization competence of male and female gametes as well as embryo development in both sea urchin species. These results are consistent with those previously reported for CNS leachate. Moreover, the characterisation of CNS leachate confirmed the presence of detectable branched polyethylenimine in the conditioned seawater even though in a very limited amount. Altogether, these data indicate that the presence of branched polyethylenimine is a cause-effect associated with a significant risk in CNS formulations due to its leaching upon contact with seawater. Nevertheless, the suggested safety protocol consisting of consecutive leaching treatments and conditioning of CNS in seawater can successfully ameliorate the CNS ecotoxicity while maintaining the efficacy of its sorbent properties supporting potential environmental applications.

An organic extract from ascidian Ciona robusta induces cytotoxic autophagy in human malignant cell lines.

The last decades have seen an increase in the isolation and characterization of anticancer compounds derived from marine organisms, especially invertebrates, and their use in clinical trials. In this regard, ascidians, which are included in the subphylum Tunicata, represent successful examples with two drugs, Aplidine© and Yondelis© that reached the market as orphan drugs against several malignancies. Here, we report that an organic extract prepared from homogenized tissues of the Mediterranean ascidian Ciona robusta inhibited cell proliferation in HT-29, HepG2, and U2OS human cells with the former being the most sensitive to the extract (EC50 = 250 µg/mL). We demonstrated that the ascidian organic extract was not cytotoxic on HT-29 cells that were induced to differentiate with sodium butyrate, suggesting a preference for the mixture for the malignant phenotype. Finally, we report that cell death induced by the organic extract was mediated by the activation of a process of cytotoxic autophagy as a result of the increased expression of the LC3-II marker and number of autophagic vacuoles, which almost doubled in the treated HT-29 cells. In summary, although the detailed chemical composition of the Ciona robusta extract is still undetermined, our data suggest the presence of bioactive compounds possessing anticancer activity.

Immune and Reproductive Biomarkers in Female Sea Urchins Paracentrotus lividus under Heat Stress.

The functioning of the immune and reproductive systems is crucial for the fitness and survival of species and is strongly influenced by the environment. To evaluate the effects of short-term heat stress (HS) on these systems, confirming and deepening previous studies, female sea urchin Paracentrotus lividus were exposed for 7 days to 17 °C, 23 and 28 °C. Several biomarkers were detected such as the ferric reducing power (FRAP), ABTS-based total antioxidant capacity (TAC-ABTS), nitric oxide metabolites (NOx), total thiol levels (TTL), myeloperoxidase (MPO) and protease (PA) activities in the coelomic fluid (CF) and mitochondrial membrane potential (MMP), H2O2 content and intracellular pH (pHi) in eggs and coelomocytes, in which TAC-ABTS and reactive nitrogen species (RNS) were also analyzed. In the sea urchins exposed to HS, CF analysis showed a decrease in FRAP levels and an increase in TAC-ABTS, TTL, MPO and PA levels; in coelomocytes, RNS, MMP and H2O2 content increased, whereas pHi decreased; in eggs, increases in MMP, H2O2 content and pHi were found. In conclusion, short-term HS leads to changes in five out of the six CF biomarkers analyzed and functional alterations in the cells involved in either reproductive or immune activities.

Nanostructured cellulose sponge engineered for marine environmental remediation: Eco-safety assessment of its leachate on sea urchin reproduction (Part A).

Nanostructured cellulose sponges (CNS) have been developed as eco-friendly and sustainable engineered materials for marine environmental remediation. Despite their functionality, sensitivity, efficiency and specificity have been proved, CNS application is still limited since their environmental safety (eco-safety) has not been completely assessed. In this study, CNS were allowed to leach in natural seawater simulating the remediation process condition and the eco-safety of CNS leachate on sea urchin reproduction has been assessed by carrying out a multi-response integrated approach, combining standardized ecotoxicity tests, innovative bioassays and gamete quality assessment. Overall, the ecotoxicity data indicate that CNS leachate affects gamete quality, gamete fertilisation competence, and embryo development probably associated with the release of chemical additives used during the synthesis process. However, in the framework of the eco-design approach, consecutive leaching treatments and conditioning of CNS in seawater open the route for a new safety protocol successfully solving the ecotoxicity while maintaining CNS sorbent properties. A safe environmental application of the resulting conditioned CNS for seawater pollution remediation is envisaged.

Electrophysiology and Fluorescence Spectroscopy Approaches for Evaluating Gamete and Embryo Functionality in Animals and Humans.

This review has examined two of the techniques most used by our research group for evaluating gamete and embryo functionality in animal species, ranging from marine invertebrates to humans. Electrophysiology has given access to fundamental information on some mechanisms underpinning the biology of reproduction. This technique demonstrates the involvement of ion channels in multiple physiological mechanisms, the achievement of homeostasis conditions, and the triggering of profound metabolic modifications, often functioning as amplification signals of cellular communication. Fluorescence spectrometry using fluorescent probes to mark specific cell structures allows detailed information to be obtained on the functional characteristics of the cell populations examined. The simple and rapid execution of this methodology allowed us to establish a panel helpful in elucidating functional features in living cells in a simultaneous and multi-parameter way in order to acquire overall drafting of gamete and embryo functionality.

Oocyte quality assessment in marine invertebrates: a novel approach by fluorescence spectroscopy.

BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is predominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

Comparison of sperm preparation methods to improve the recovery of mature spermatozoa in sub-fertile males.

The integrity of chromatin in the spermatozoon is essential for reproductive outcome. The aim of this study was to evaluate the most effective and cost-effective method to reduce the percentage of spermatozoa with defects in chromatin decondensation for use in assisted reproductive technologies (ART) procedures. Sperm samples from 15 sub-fertile males were examined at CFA Naples to determine the sperm decondensation index (SDI), using the aniline blue test, before and after preparation, comparing density gradients with two different swim-up approaches. All three techniques led to a reduction in decondensed spermatozoa with no statistical difference (P > 0.05) between the control and the treated sperm. In contrast, we found a highly significant decrease in SDI (P < 0.01) after the two swim-up methods in all the samples, confirming the efficacy of these methods in lowering the percentage of chromatin compaction damage. There was no statistical difference between the two swim-up methods, however swim-up from the pellet led to improved count, motility and the percentage of normal condensed spermatozoa. We suggest that swim-up from the pellet be used in ART on sub-fertile males, both to reduce cell stress by multiple centrifugation and improve the recovery rate of mature spermatozoa.

Pathophysiological Responses to Conotoxin Modulation of Voltage-Gated Ion Currents.

Voltage-gated ion channels are plasma membrane proteins that generate electrical signals following a change in the membrane voltage. Since they are involved in several physiological processes, their dysfunction may be responsible for a series of diseases and pain states particularly related to neuronal and muscular systems. It is well established for decades that bioactive peptides isolated from venoms of marine mollusks belonging to the Conus genus, collectively known as conotoxins, can target different types and isoforms of these channels exerting therapeutic effects and pain relief. For this reason, conotoxins are widely used for either therapeutic purposes or studies on ion channel mechanisms of action disclosure. In addition their positive property, however, conotoxins may generate pathological states through similar ion channel modulation. In this narrative review, we provide pieces of evidence on the pathophysiological impacts that different members of conotoxin families exert by targeting the three most important voltage-gated channels, such as sodium, calcium, and potassium, involved in cellular processes.

Oocyte quality assessment in marine invertebrates: a novel approach by fluorescence spectroscopy

BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is pre-dominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

Sperm Motility, Oxidative Status, and Mitochondrial Activity: Exploring Correlation in Different Species.

Sperm quality assessment is the first step for evaluating male fertility and includes the estimation of sperm concentration, motility, and morphology. Nevertheless, other parameters can be assessed providing additional information on the male reproductive potential. This study aimed to evaluate and correlate the oxidative status, mitochondrial functionality, and motility in spermatozoa of two marine invertebrate (Ciona robusta and Mytilus galloprovincialis) and one mammalian (Bos taurus) species. By combining fluorescent staining and spectrofluorometer, sperm oxidative status was evaluated through intracellular reactive oxygen species (ROS) and plasma membrane lipid peroxidation (LPO) analysis. Mitochondrial functionality was assessed through the mitochondrial membrane potential (MMP). In the three examined species, a negative correlation emerged between sperm motility vs ROS levels and LPO. Sperm motility positively correlated with MMP in bovine, whereas these parameters were not related in ascidian or even negatively related in mussel spermatozoa. MMP was negatively related to ROS and LPO levels in ascidians, only to LPO in bovine, and positively related in mussel spermatozoa. These results suggest that energy sources for sperm motility vary between species and that ROS causes a decline in sperm motility via oxidative damage of membrane lipids. Overall, this study validates the use of fluorescent probes in combination with spectrofluorometer as a simple and powerful methodology for supplementary evaluation of sperm quality shedding light on new potential quality markers and provided relevant information on sperm energetic metabolism.

The Era of Nanomaterials: A Safe Solution or a Risk for Marine Environmental Pollution?

In recent years, the application of engineered nanomaterials (ENMs) in environmental remediation gained increasing attention. Due to their large surface area and high reactivity, ENMs offer the potential for the efficient removal of pollutants from environmental matrices with better performances compared to conventional techniques. However, their fate and safety upon environmental application, which can be associated with their release into the environment, are largely unknown. It is essential to develop systems that can predict ENM interactions with biological systems, their overall environmental and human health impact. Until now, Life-Cycle Assessment (LCA) tools have been employed to investigate ENMs potential environmental impact, from raw material production, design and to their final disposal. However, LCA studies focused on the environmental impact of the production phase lacking information on their environmental impact deriving from in situ employment. A recently developed eco-design framework aimed to fill this knowledge gap by using ecotoxicological tools that allow the assessment of potential hazards posed by ENMs to natural ecosystems and wildlife. In the present review, we illustrate the development of the eco-design framework and review the application of ecotoxicology as a valuable strategy to develop ecosafe ENMs for environmental remediation. Furthermore, we critically describe the currently available ENMs for marine environment remediation and discuss their pros and cons in safe environmental applications together with the need to balance benefits and risks promoting an environmentally safe nanoremediation (ecosafe) for the future.

Neurobiological activity of conotoxins via sodium channel modulation.

Conotoxins (CnTX) are bioactive peptides produced by marine molluscs belonging to Conus genus. The biochemical structure of these venomous peptides is characterized by a low number of amino acids linked with disulfide bonds formed by a high degree of post-translational modifications and glycosylation steps which increase the diversity and rate of evolution of these molecules. CnTX different isoforms are known to target ion channels and, in particular, voltage-gated sodium (Na+) channels (Nav channels). These are transmembrane proteins fundamental in excitable cells for generating the depolarization of plasma membrane potential known as action potential which propagates electrical signals in muscles and nerves for physiological functions. Disorders in Nav channel activity have been shown to induce neurological pathologies and pain states. Here, we describe the current knowledge of CnTX isoform modulation of the Nav channel activity, the mechanism of action and the potential therapeutic use of these toxins in counteracting neurological dysfunctions.

Integrated characterization and risk management of marine sediments: The case study of the industrialized Bagnoli area (Naples, Italy).

The aim of the present work is to demonstrate the practical importance of a multidisciplinary approach and weighted criteria to synthesize and integrate different typologies of data (or lines of evidence, LOEs), including chemical levels in marine sediments, their bioavailability to specific indicator species, ecotoxicological effects measured through subcellular biomarkers and batteries of bioassays, and potential impacts of pollution on local benthic communities. The area of Bagnoli (Gulf of Naples, Southern Italy) was selected as a model case-study, as it is a coastal area chronically impacted by massive industrial contamination (trace metals and hydrocarbons), and dismissed decades ago without any subsequent remediation or habitat restoration. The results of each LOE were elaborated to provide specific hazard indices before their overall integration in a weight of evidence (WOE) evaluation. Levels of some trace metals and PAHs revealed a severe contamination in the entire study area. Bioavailability of hydrocarbons was evident particularly for high molecular weight PAHs, which also caused significant variations of cellular biomarkers, such as cytochrome P450 metabolization in fish, lysosomal membrane destabilization in mussels, genotoxic effects both in fish and molluscs. The results of a battery of bioassays indicated less marked responses compared to those obtained from chemical and biomarkers analyses, with acute toxicity still present in sediments close to the source of contamination. The analysis of benthic assemblages showed limited evidence of impact in the whole area, indicating a good functioning of local ecosystems at chronic contamination. Overall, the results of this study confirm the need of combining chemical and biological data, the quantitative characterization of various typologies of hazard and the importance of assessing an integrated environmental WOE risk, to orientate specific and scientifically-supported management options in industrialized areas.

Adult exposure to acidified seawater influences sperm physiology in Mytilus galloprovincialis: Laboratory and in situ transplant experiments.

The ongoing increase of CO2 in the atmosphere is inducing a progressive lowering of marine water pH that is predicted to decrease to 7.8 by the end of this century. In marine environment, physical perturbation may affect reproduction, which is crucial for species' survival and strictly depends on gamete quality. The effects of seawater acidification (SWAc) on gamete quality of broadcast spawning marine invertebrates result largely from experiments of gamete exposure while the SWAc impact in response to adult exposure is poorly investigated. Performing microcosm and in field experiments at a naturally acidified site, we investigated the effects of adult SWAc exposure on sperm quality parameters underlying fertilization in Mytilus galloprovincialis. These animals were exposed to pH 7.8 over 21 days and collected at different times to analyze sperm parameters as concentration, motility, viability, morphology, oxidative status, intra- and extra-cellular pH and mitochondrial membrane potential. Results obtained in the two experimental approaches were slightly different. Under field conditions, we found an increase in total sperm motility and mitochondrial membrane potential on days 7 and 14 from the start of SWAc exposure whereas, in microcosm, SWAc group showed an increase of total motility on day 14. In addition, sperm morphology and intracellular pH were affected in both experimental approaches; whereas oxidative stress was detected only in spermatozoa collected from mussels under natural SWAc. The overall analysis suggests that, in mussels, SWAc toxic mechanism in spermatozoa does not involve oxidative stress. This study represents the first report on mussel sperm quality impairment after adult SWAc exposure, which may affect fertilization success with negative ecological and economic consequences; it also indicates that, although naturally acidified areas represent ideal natural laboratories for investigating the impact of ocean acidification, microcosm experiments are necessary for examining action mechanisms.

Gamete quality in a multistressor environment.

Over the past few decades, accumulated evidence confirms that the global environment conditions are changing rapidly. Urban industrialization, agriculture and globalization have generated water, air and soil pollution, giving rise to an environment with a growing number of stress factors, which has a serious impact on the fitness, reproduction and survival of living organisms. The issue raises considerable concern on biodiversity conservation, which is now at risk: it is estimated that a number of species will be extinct in the near future. Sexual reproduction is the process that allows the formation of a new individual and is underpinned by gamete quality defined as the ability of spermatozoa and oocytes to interact during fertilization leading to the creation and development of a normal embryo. This review aimed to provide the current state of knowledge regarding the impact of a broad spectrum of environmental stressors on diverse parameters used to estimate and evaluate gamete quality in humans and in canonical animal models used for experimental research. Effects of metals, biocides, herbicides, nanoparticles, plastics, temperature rise, ocean acidification, air pollution and lifestyle on the physiological parameters that underlie gamete fertilization competence are described supporting the concept that environmental stressors represent a serious hazard to gamete quality with reproductive disorders and living organism failure. Although clear evidence is still limited, gamete capacity to maintain and/or recover physiological conditions is recently demonstrated providing further clues about the plasticity of organisms and their tolerance to the pressures of pollution that may facilitate the reproduction and the persistence of species within the scenario of global change. Changes in the global environment must be urgently placed at the forefront of public attention, with a massive effort invested in further studies aimed towards implementing current knowledge and identifying new methodologies and markers to predict impairment of gamete quality.

Ocean acidification impact on ascidian Ciona robusta spermatozoa: New evidence for stress resilience.

Rising atmospheric CO2 is causing a progressive decrease of seawater pH, termed ocean acidification. Predicting its impact on marine invertebrate reproduction is essential to anticipate the consequences of future climate change on species fitness and survival. Ocean acidification may affect reproductive success either in terms of gamete or progeny quality threating species survival. Despite an increasing number of studies focusing on the effects of ocean acidification on the early life history of marine organisms, very few have investigated the effects on invertebrate gamete quality. In this study, we set up two experimental approaches simulating the ocean conditions predicted for the end of this century, in situ transplant experiments at a naturally acidified volcanic vent area along the Ischia island coast and microcosm experiments, to evaluate the short-term effects of the predicted near-future levels of ocean acidification on sperm quality of the ascidian Ciona robusta after parental exposure. In the first days of exposure to acidified conditions, we detected alteration of sperm motility, morphology and physiology, followed by a rapid recovery of physiological conditions that provide a new evidence of resilience of ascidian spermatozoa in response to ocean acidification. Overall, the short-term tolerance to adverse conditions opens a new scenario on the marine species capacity to continue to reproduce and persist in changing oceans.

Cytotoxicity and genotoxicity of CuO nanoparticles in sea urchin spermatozoa through oxidative stress.

Copper oxide nanoparticles (CuO NPs) are extensively used in various industrial and commercial applications. Despite their wide application may lead to the contamination of marine ecosystem, their potential environmental effects remain to be determined. Toxicity assessment studies have primarily focused on investigating the effects of CuO NPs on fertilization success and embryo development of different sea urchin species while the impact on sperm quality have never been assessed. In this line, this study aims to assess the effects of CuO NPs on the spermatozoa of the sea urchin Paracentrotus lividus. After sperm exposure to CuO NPs, biomarkers of sperm viability, cytotoxicity, oxidative stress, and genotoxicity as well as morphology were evaluated. Results showed that CuO NPs exposure decreased sperm viability, impaired mitochondrial activity and increased the production of reactive oxygen species (ROS) and lipid peroxidation. Furthermore, CuO NPs exposure caused DNA damage and morphological alterations. Together with the antioxidant rescue experiments, these results suggest that oxidative stress is the main driver of CuO NP spermiotoxic effects. The mechanism of toxicity is here proposed: the spontaneous generation of ROS induced by CuO NPs and the disruption of the mitochondrial respiratory chain lead to production of ROS that, in turn, induce lipid peroxidation and DNA damage, and result in defective spermatozoa up to induce sperm cytotoxicity. Investigating the effects of CuO NPs on sea urchin spermatozoa, this study provides valuable insights into the mechanism of reproductive toxicity induced by CuO NPs.

Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact.

The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity.